Novelty in improvement of CAR T cell-based immunotherapy with the aid of CRISPR system

Abstract Introduction Chimeric Antigen Receptor (CAR) T cells have tremendous potentials for cancer treatment; however, various challenges impede their universal use. These restrictions include the poor function of T cells in tumor microenvironments, the shortage of tumor-specific antigens and, finally, the high cost and time-consuming process, as well as the poor scalability of the method. Creative gene-editing tools have addressed each of these limitations and introduced next generation products for cell therapy. The clustered regularly interspaced short palindromic repeats-associated endonuclease 9 (CRISPR/Cas9) system has triggered a revolution in biology fields, as it has a great capacity for genetic manipulation. Method In this review, we considered the latest development of CRISPR/Cas9 methods for the chimeric antigen receptor T cell (CAR T)-based immunotherapy. Results The ability of the CRISPR/Cas9 system to generate the universal CAR T cells and also potent T cells that are persistent against exhaustion and inhibition was explored. Conclusion: We explained CRISPR delivery methods, as well as addressing safety concerns related to the use of the CRISPR/Cas9 system and their potential solutions.

Construction of HIF-1α gene knockout plasmid and functional verification in naked mole rats based on CRISPR/Cas9 system / 中国实验动物学报

Objective To construct plasmids and knock out HIF-1α gene expression in an naked mole rat skin fibroblasts(NSF)cell line using CRISPR/Cas9 genomic editing technology,to provide an in vitro cell model for studying the mechanism of hypoxia tolerance and the occurrence and development of hypoxia-related diseases in naked mole rats.Methods We designed four pairs of single guide RNA(sgRNA)sequences targeting exons 1～4 of the NSF HIF-1αgene and successfully constructed an expression plasmid.The plasmid with the optimal sgRNA was identified and transfected into 293T cells,and the supernatant was used for detecting the virus titer.Lentivirus particles carrying sgRNAs of HIF-1α were transfected into NSF cells which express Cas9 protein,based on a previous protocol.After transfection,fluorescence signals were observed under a fluorescence microscope,and HIF-1α expression in NSF cells was detected by Western Blot and T7 endonuclease 1(T7E1)analysis.Results Sanger sequencing showed that the designed sgRNA was successfully inserted into pX459 and pKLV2-U6-sgRNA2 vectors,demonstrating successful construction of a recombinant plasmid for transfection.T7E1 digestion successfully removed three bands and the target efficiency of sgRNA was 54％.Western Blot showed that the HIF-1α gene was successfully knocked out and its protein level was significantly reduced in NSF cells from naked mole rats(P=0.0019).There were no obvious morphological changes in HIF-1α-knockout cells under the microscope,and gene knockout had no obvious effect on cell proliferation.Conclusions We successfully constructed an HIF-1α-knockout cell line using CRISPR/Cas9 technology,to provide an experimental basis for further studies of the biological function of HIF-1α,as well as the mechanism of hypoxia tolerance in naked mole rats.The result also provide a theoretical foundation for the prevention and treatment of hypoxia-related diseases.

Construction of Dmd Gene Mutant Mice and Phenotype Verification in Muscle and Immune Systems / 实验动物与比较医学

Objective The aim is to utilize CRISPR/Cas9 gene editing technology to construct Dmd gene mutant mice with a point mutation in exon 23 of the Dmd gene. Subsequently, the phenotypic changes of the mice in muscles and immune systems are analyzed and verified, providing an evaluation model for Duchenne muscular dystrophy and other related diseases.MethodsBased on the sequence characteristics of exon 23 of the Dmd gene, small guide RNA (sgRNA) was designed and synthesized. Cas9 mRNA, sgRNA fragments, and oligo donor DNA were microinjected into fertilized eggs of C57BL/6J mice. After transferring the fertilized eggs to surrogate mice, F0 generation mice were born. After mating with F0 generation mice, offspring mice were obtained, and Dmd gene positive mutant (DmdMu/+) mice were obtained after genotype identification. Male hemizygous DmdMu/+(DmdMu/Y) mice were selected for phenotype validation. The body weight of live 3- and 9-month-old mice were recorded. Muscle tension was evaluated through the grid test. Hearts and semitendinosus muscles were collected, and the histopathological changes were observed using HE staining. Further, the expression of Dmd protein in muscle tissue of 9-month-old mice was analyzed by Western blotting.An acute inflammation model was established in DmdMu/Y mice using lipopolysaccharide induction. Peripheral blood from the submandibular vein was collected, and the changes in the proportion of neutrophils and monocytes were detected by flow cytometry.Results The results of genome sequencing and Western blotting confirmed the successful construction of Dmd gene point mutant mice (DmdMu/+ mice). Dmd protein expression was not detected in skeletal muscle and myocardium of DmdMu/+ mice, and it was significantly reduced compared to wild-type C57BL/6J mice (P<0.05). Compared with wild-type mice of the same background, DmdMu/Y mice at 3 and 9 months of age showed significant weight loss (P<0.01) and decreased muscle tension (P<0.05). 9-month-old DmdMu/Y mice exhibited significant pathological changes in skeletal muscle and myocardium, including widening of intermuscular space. Under normal condition, compared with wild-type mice, the proportion of neutrophils and monocytes in the peripheral blood of 3-month-old DmdMu/Y mice was significantly lower than that of wild-type mice (P<0.01). After lipopolysaccharide stimulation, the proportion of neutrophils in peripheral blood of 3-month-old DmdMu/Y mice remained significantly lower compared to that of wild-type mice (P<0.01). The proportion of neutrophils in peripheral blood of 9-month-old DmdMu/Y mice significantly decreased after lipopolysaccharide induction (P<0.01), with a trend of change observed in monocytes between groups.Conclusion The successful construction of the Dmd gene mutant mouse model has confirmed the vital function of Dmd gene in maintaining normal muscle tissue morphology and muscle tone. It preliminarily indicated that Dmd gene deletion could significantly reduce the proportion of neutrophils in peripheral blood, offering a new perspective for the study of immune system alterations in Duchenne muscular dystrophy patients.

Application of CRISPR/Cas9 gene editing technology in the research of precision oncology / 中国肿瘤生物治疗杂志

@#[摘 要] CRISPR/Cas9基因编辑技术通过精准定位和修改基因序列，可以识别与细胞增殖、迁移、侵袭和化疗耐药性相关的基因，不仅为理解肿瘤发生发展的分子机制奠定了基础，还为实现肿瘤的精准治疗提供了一种方便、高效的方法。由于其具有低成本、高效率的优点，被广泛地应用于精准肿瘤学的基础和临床研究当中，包括用于探寻抗肿瘤药物耐药靶点、筛查驱动基因、优化CAR-T和TCR-T细胞，以及筛选肿瘤靶向基因等。目前，已开展了十余项项使用CRISPR/Cas9技术治疗肿瘤的临床试验，策略多为利用CRISPR/Cas9技术敲除T细胞中的免疫检查点基因后回输患者，以达到免疫激活的效果，大多数研究仍处于Ⅰ期和Ⅱ期阶段。管CRISPR/Cas9基因编辑技术在肿瘤研究与治疗领域展现出了巨大潜力，但仍需面对脱靶效应，以及永久编辑可能带来的弊端等瓶颈，其实际临床效用仍有待更多的深入研究和大规模临床试验的严格验证。

Construction and gene identification of myeloid specific Spi1 knockout mice / 安徽医科大学学报

Objective @#To construct myeloid specific Spi1 gene knockout mice and analyze their genotypes , so as to provide animal model basis for the study of pathological mechanism of diseases and drug targets .@*Methods @#According to the principle of CRISPR/Cas9 technology and C re/LoxP system , sgRNA and Donor vectors were de signed and constructed . The transcript of Exon 2 ( Exon 2) was used as the knockout region , and Loxp elements were placed on both sides of Exon 2 . Cas9 protein , sgRNA and Donor vector were mixed and microinj ected into the fertilized eggs of C57BL/6J mice , the fertilized eggs were transplanted into the uterus of C57BL/6J pregnant female mice , and F0 generation was obtained after 19 ~ 20 days . Positive F0 mice were mated with C57BL/6J mice to ob tain stable F1 Spi1 flox/ + mice . Spi1 flox/ + mice of F1 generation were selfed to obtain Spi1 flox/flox mice . Spi1 flox/flox mated with Lyz2-Cre + mice to obtain Spi1 flox/ + /Lyz2-Cre + mice , and then mated with Spi1 flox/flox , the Spi1 flox/flox/Lyz2-Cre + mice were myeloid specific Spi1 gene knockout ( KO) mice . Spi1 flox/flox/Lyz2-cre - mice were used as wild type (WT) mice . DNA of WT and KO mice was extracted , and the genotypes were identified by agarose gel electro phoresis after PCR amplification . Western blot was used to detect the expression of spleen focus forming virus proviral integration oncogene , Spi - 1 /purine rich box - 1(PU . 1) in immune cells of WT and KO mice .@*Results@#The results of PCR identification showed that the genotype of mice with only 220 bp amplified by flox primer was Spi1 flox/flox homozygote , and the genotype of mice with 700 bp amplified by Lyz2-Cre primer was Lyz2-Cre + . Western blot showed that compared with WT group , the protein PU . 1 was not expressed in bone marrow derived macropha ges (BMDMs ) and peritoneal macrophages (PM) in KO group (P < 0.01) . There was no significant difference of statistics in the expression level of PU . 1 in T cells between KO mice and WT mice . The results of PCR and West ern blot showed that myeloid specific Spi1 KO mice were successfully constructed . @*Conclusion @#The myeloid spe cific Spi1 gene KO mice are successfully constructed and identified , which provides animal model basis for further revealing the potential mechanism of PU . 1 inimmune regulation .

Construction of Raji-Luc CD19 KO Lymphoma Cell Line Using CRISPR/Cas9 Technology / 现代检验医学杂志

Objective To construct Raji-Luc lymphoma cells with CD19 knockout using CRISPR/Cas9 technology and preliminarily validate their immune escape ability.Methods PB-CRISPR-CD19 small guide RNA(sgRNA)plasmids was constructed,the optimal sgRNA sequence was screened,and Raji-Luc cells with pCAG-PBase,PB-CD19 sgRNA,and PB-CRISPR-Cas9 were co-transfected.Stable knockout monoclonal cell lines were screened by flow sorting and limit dilution method and the knockout effect was verified through gene sequence testing.The expression of luciferase on the surface of the cell line was detected by microplate reader,CD19 CAR-T and CD38 CAR-T previously constructed in the laboratory were used as effector cells,and the immune escape ability of Raji-Luc CD19 KO cell line was verified by universal luciferase chemiluminescence method.Results The transfection efficiency of Raji-Luc CD19 KO cells prepared by electro transfection was high,and the knockout efficiency of the two monoclonal cells was more than 99%.There was no significant difference in luciferase expression compared to the original Raji-Luc cells,and CD19 CAR-T cells could not be activated to the kill them.Conclusion Successfully constructed Raji-Luc CD19 KO lymphoma cell line.

Phosphatidylinositol 3-kinases Play a Suppressive Role in Electrotaxis of Dictyostelium Cells Through Akt and ERK / 生物化学与生物物理进展

ObjectivePhosphatidylinositol 3 kinases (PI3Ks) play an important role in cell directional movement by regulating F-actin. However, the structure and function of PI3Ks are complex. The role of PI3Ks in cell electrotaxis is not fully understood. Therefore, in this study, the model organism Dictyostelium discoideum cells were used as experimental materials to explore the role of PI3K1 and PI3K2 in electrotaxis. MethodsFirstly, PI3K1 coding gene pikA knockout mutant and PI3K2 coding gene pikB knockout mutant were constructed by CRISPR/Cas9 system. Secondly, two mutants were placed in a DC electric field with a strength of 12 V/cm and the electrotaxis were analyzed. ResultsData analysis showed that the direction index of wild-type cells in DC electric field was (0.86±0.03), while the direction index of pikA- and pikB- mutants in DC electric field was (0.95±0.02) and (0.94±0.03), respectively. In addition, the average trajectory speed of wild-type cells in the electric field was (3.34±0.08) μm/min, while the average trajectory speed of pikA- and pikB- mutants were (4.85±0.20) μm/min and (5.48±0.15) μm/min, respectively. The t test showed that there were significant differences in the directedness index and speed between the mutant and wild type. Western blot results showed that both phosphorylated Akt and phosphorylated ERK were significantly increased in pikA- and pikB- mutants. ConclusionPI3K1 and PI3K2 may inhibit the electrotaxis of Dictyostelium discoideum cells by increasing the activity of Akt and ERK.

Breeding and genotyping of T lymphocyte-conditional Spi1 knockout mice / 安徽医科大学学报

Objective @#To breed and identify the T lymphocyte-conditional Spi1 knockout mice for the further in- vestgation of the specific role of Spi1-encoded protein PU. 1 . @*Methods @#The Lck-Cre mice were mated with Spi1 flox/flox mice to obtain Lck-Cre ×Spi1 flox/flox mice (T lymphocyte-specific Spi1 knockout mice) , and the genotype was determined by polymerase chain reaction (PCR) and agarose gel electrophoresis . Magnetic beads were used to sort out the splenic T lymphocytes , and the knockdown efficiency of PU. 1 in T cells was detected by Western blot , quantitative real-time PCR ( qPCR) and flow cytometry. @*Results @#The Lck-Cre ×Spi1 flox/flox mouse genotype was stably inherited . Compared with Spi1 flox/flox mice , the expression level of PU. 1 was significantly reduced in splenic T cells of Lck-Cre ×Spi1 flox/flox mice . @*Conclusion @#In this study , the T lymphocyte-specific Spi1 knockout mice was successfully constructed by applying Cre/LoxP system and CRISPR/Cas9 technology , which provided a reliable an- imal model for the subsequent experiments of the specific role of PU. 1 in T cell-related diseases .

Novel approaches for HTLV-1 therapy: innovative applications of CRISPR-Cas9

ABSTRACT The human T-cell lymphotropic virus type 1 (HTLV-1) is a single-stranded positive-sense RNA virus that belongs to the Retroviridae family, genus Deltaretro, and infects approximately five to 10 million people worldwide. Although a significant number of individuals living with HTLV-1 remain asymptomatic throughout their lives, some develop one or more severe clinical conditions, such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a progressive and debilitating disease, and/or a subtype of non-Hodgkin's lymphoma with a more threatening course known as adult T-cell leukemia/lymphoma (ATLL). Moreover, current therapeutic options are limited and focus primarily on treating symptoms and controlling viral latency. CRISPR-Cas9 gene editing is proposed as a promising tool to address the intricate links associated with HTLV-1. By targeting or silencing key genes during initial infection and dysregulating immune signaling pathways, CRISPR-Cas9 offers potential intervention opportunities. In this review, we address the therapeutic potential of CRISPR-Cas9 gene editing, as well as examine the primary mechanisms involved in editing potential target genes and discuss the existing evidence in the current scientific literature.

Construction of interferon alpha/beta receptor subunit 1 gene knockout Caco-2 cell line based on CRISPR/Cas9 system / 中国生物制品学杂志

@#Objective To knockout interferon alpha/beta receptor subunit 1(IFNAR1) gene in human colorectal adenocarcinoma cells Caco-2 using clustered regularly interspaced short palinmic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)system to construct IFNAR1 knockout Caco-2 cell line.Methods The single guide RNA(sgRNA)sequence was designed to specifically recognize the exon region of IFNAR1 gene using CRISPR/Cas9 technology,and the LentiCRISPRv2-IFNAR1-sgRNA recombinant plasmid was constructed.Caco-2 cells were infected with the plasmid packaged by lentivirus and screened by puromycin resistance.The obtained monoclonal cell lines were cultured by limited dilution method,which were verified for the effect of IFNAR1 gene knockout by target gene sequencing and Western blot,and detected for the mRNA levels of CXC chemokine ligand 10(CXCL10)and interferon-stimulatd gene 20(ISG20)in IFNAR1knockout cells by adding exogenous IFNβ.Results Sequencing results of plasmid LentiCRISPRv2-IFNAR1-sgRNA showed that the insertion sites were all located at the sticky end of BsmBⅠenzyme digestion.Two IFNAR1 knockout monoclonal cell lines were obtained.The sequencing results showed that Caco-2-IFNAR1-KO1 had 5 bp deletion in the sixth exon of IFNAR1,and Caco-2-IFNAR1-KO2 had 18 bp deletion and 1 bp insertion in the seventh exon.Compared with wild-type Caco-2 cells,Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells showed no expression of IFNAR1 protein.Compared with no IFNβ stimulation,the mRNA levels of CXCL10 gene(t = 0.566 and 1.268 respectively,P>0.05)and ISG20 gene(t =1.522 and 1.733 respectively,P>0.05)in Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells stimulated by 50 ng/mL IFNβ showed no significant increase.While compared with those of wild-type Caco-2 cells,the mRNA levels of CXCL10gene(t = 6.763 and 6.777 respectively,P<0.05)and ISG20 gene(t = 5.664 and 5.65 respectively,P<0.05)in Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells decreased significantly under the stimulation of 50 ng/mL exogenous IFNβ.Conclusion Caco-2 cell line with IFNAR1 knockout was successfully constructed by using CRISPR/Cas9 technology,and the downstream molecules activated by IFNAR(interferon alpha/beta receptor)in this cell line were obviously inhibited,which provided a powerful tool for further exploration of the innate immune response and replication packaging mechanism of Caco-2 cells after virus infection.

Development of porcine induced pluripotent stem cells with a CD163 reporter system / 生物工程学报

As main recipient cells for porcine reproductive and respiratory syndrome virus (PRRSV), porcine alveolar macrophage (PAM) are involved in the progress of several highly pathogenic virus infections. However, due to the fact that the PAM cells can only be obtained from primary tissues, research on PAM-based virus-host interactions remains challenging. The improvement of induced pluripotent stem cells (iPSCs) technology provides a new strategy to develop IPSCs-derived PAM cells. Since the CD163 is a macrophage-specific marker and a validated receptor essential for PRRSV infection, generation of stable porcine induced pluripotent stem cells lines containing CD163 reporter system play important roles in the investigation of IPSCs-PAM transition and PAM-based virus-host interaction. Based on the CRISPR/Cas9- mediated gene editing system, we designed a sgRNA targeting CD163 locus and constructed the corresponding donor vectors. To test whether this reporter system has the expected function, the reporter system was introduced into primary PAM cells to detect the expression of RFP. To validate the low effect on stem cell pluripotency, we generated porcine iPSC lines containing CD163 reporter and assessed the pluripotency through multiple assays such as alkaline phosphatase staining, immunofluorescent staining, and EdU staining. The red-fluorescent protein (RFP) expression was detected in CD163-edited PAM cells, suggesting that our reporter system indeed has the ability to reflect the expression of gene CD163. Compared with wild-type (WT) iPSCs, the CD163 reporter-iPSCs display similar pluripotency-associated transcription factors expression. Besides, cells with the reporter system showed consistent cell morphology and proliferation ability as compared to WT iPSCs, indicating that the edited-cells have no effect on stem cell pluripotency. In conclusion, we generated porcine iPSCs that contain a CD163 reporter system. Our results demonstrated that this reporter system was functional and safe. This study provides a platform to investigate the iPS-PAM development and virus-host interaction in PAM cells.

Identification of the target site of antimicrobial peptide AMP-17 against Candida albicans / 生物工程学报

Candida albicans is one of the major causes of invasive fungal infections and a serious opportunistic pathogen in immunocompromised individuals. The antimicrobial peptide AMP-17 has prominent anti-Candida activity, and proteomic analysis revealed significant differences in the expression of cell wall (XOG1) and oxidative stress (SRR1) genes upon the action of AMP-17 on C. albicans, suggesting that AMP-17 may exert anti-C. albicans effects by affecting the expression of XOG1 and SRR1 genes. To further investigate whether XOG1 and SRR1 genes were the targets of AMP-17, C. albicans xog1Δ/Δ and srr1Δ/Δ mutants were constructed using the clustered regulatory interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) system. Phenotypic observations revealed that deletion of two genes had no significant effect on C. albicans growth and biofilm formation, whereas XOG1 gene deletion affected in vitro stress response and mycelium formation of C. albicans. Drug sensitivity assay showed that the MIC80 values of AMP-17 against xog1Δ/Δ and srr1Δ/Δ mutants increased from 8 μg/mL (for the wild type C. albicans SC5314) to 16 μg/mL, while the MIC80 values against srr1Δ/Δ: : srr1 revertants decreased to the level of the wild type SC5314. In addition, the ability of AMP-17 to inhibit biofilm formation of both deletion strains was significantly reduced compared to that of wild type SC5314, indicating that the susceptibility of the deletion mutants to AMP-17 was reduced in both the yeast state and during biofilm formation. These results suggest that XOG1 and SRR1 genes are likely two of the potential targets for AMP-17 to exert anti-C. albicans effects, which may facilitate further exploration of the antibacterial mechanism of novel peptide antifungal drugs.

Application of CRISPR-Cas9 gene editing technology in crop breeding / 生物工程学报

The CRISPR-Cas9 system is composed of a clustered regularly interspaced short palindromic repeat (CRISPR) and its associated proteins, which are widely present in bacteria and archaea, serving as a specific immune protection against viral and phage secondary infections. CRISPR-Cas9 technology is the third generation of targeted genome editing technologies following zinc finger nucleases (ZFNs) and transcription activator like effector nucleases (TALENs). The CRISPR-Cas9 technology is now widely used in various fields. Firstly, this article introduces the generation, working mechanism and advantages of CRISPR-Cas9 technology; secondly, it reviews the applications of CRISPR-Cas9 technology in gene knockout, gene knock-in, gene regulation and genome in breeding and domestication of important food crops such as rice, wheat, maize, soybean and potato. Finally, the article summarizes the current problems and challenges encountered by CRISPR-Cas9 technology and prospects future development and application of CRISPR-Cas9 technology.

CRISPR/Cas9-mediated endogenous TCR knockout enhances TCR-T cells targeted killing HPV16-positive cervical cancer cells / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：基于CRISPR/Cas9基因编辑技术制备无内源TCR的TCR-T细胞并鉴定其在体外杀伤HPV16阳性宫颈癌SiHa细胞的功能。方法：培养健康志愿者外周血CD8+ T细胞和Jurkat细胞，CRISPR/Cas9基因编辑技术敲除CD8+ T、Jurkat细胞的TCR基因，制备过表达转基因TCR的重组慢病毒，在敲除内源性TCR的CD8+ T和Jurkat细胞中用慢病毒过表达转基因TCR制备TCR-T细胞，多色FCM检测TCR-T细胞中TCR和CD3的表达水平，荧光素酶活性实验检测TCR-T细胞对HPV16阳性SiHa细胞的杀伤效率。结果：CRIPSR/Cas9基因编辑技术高效地敲除了外周血CD8+ T细胞和Jurkat细胞中的TRAC和TRBC基因，敲除效率分别为（81.4±4.5）%、（98.5±0.07）%，制备的无内源TCR的TCR-T细胞高效表达转基因TCR，在外周血CD8+ T和Jurkat细胞中表达率为（66.0±17.8）%、（97.3±2.6）%，敲除内源TRAC和TRBC基因有效增强CD8+ T和Jurkat细胞膜表达转基因TCR（均P<0.01），敲除内源TCR增强TCR-T细胞特异性杀伤HPV16阳性的SiHa细胞[（71.4±1.0）% vs （35.1±2.0）%，P<0.01）]。结论：无内源TCR的TCR-T细胞显著增强转基因TCR的表达和对HPV16阳性宫颈癌SiHa细胞的靶向杀伤能力，为提高TCR-T细胞的临床疗效提供了实验依据。

Construction of CRISPR/Cas9 system targeting mouse Tsc1 and Tsc2 gene and verification of targeting effectiveness / 中国生物制品学杂志

@#ObjectiveTo design and construct CRISPR/Cas9 gene editing system targeting Tsc1 and Tsc2 genes,and verify the effectiveness of gene editing at cellular level.MethodsThree sgRNA guide sequences were designed for mouse Tsc1 and Tsc2 genes respectively. The sgRNA expression vector was constructed and co-transfected with the Cas9 expression plasmid into mouse N2a cells. After the positive cells were obtained through drug screening,the DNA fragments at the targeting site were amplified by PCR,and the targeting efficiency was verified by TA clone sequencing.ResultsThe five targets of Tsc1-M-sgRNA2 and Tsc1-M-sgRNA3 of Tsc1 gene and Tsc2-M-sgRNA1,Tsc2-M-sgRNA2 and Tsc2-M-sgRNA3 of Tsc2 gene were all edited,and the editing efficiency was 40%,80%,30%,30% and 20%,respectively.ConclusionA CRISPR-Cas9 gene editing system with editing efficiency targeting mouse Tsc1 and Tsc2 genes was successfully constructed.

Generation of Mlk3 KO mice by CRISPR/Cas9 and its effect on blood pressure / 生物工程学报

To explore the effect of Mlk3 (mixed lineage kinase 3) deficiency on blood pressure, Mlk3 gene knockout (Mlk3KO) mice were generated. Activities of sgRNAs targeted Mlk3 gene were evaluated by T7 endonuclease I (T7E1) assay. CRISPR/Cas9 mRNA and sgRNA were obtained by in vitro transcription, microinjected into zygote, followed by transferring into a foster mother. Genotyping and DNA sequencing confirmed the deletion of Mlk3 gene. Real- time PCR (RT-PCR), Western blotting or immunofluorescence analysis showed that Mlk3KO mice had an undetectable expression of Mlk3 mRNA or Mlk3 protein. Mlk3KO mice exhibited an elevated systolic blood pressure compared with wild-type mice as measured by tail-cuff system. Immunohistochemistry and Western blotting analysis showed that the phosphorylation of MLC (myosin light chain) was significantly increased in aorta isolated from Mlk3KO mice. Together, Mlk3KO mice was successfully generated by CRISPR/Cas9 system. MLK3 functions in maintaining blood pressure homeostasis by regulating MLC phosphorylation. This study provides an animal model for exploring the mechanism by which Mlk3 protects against the development of hypertension and hypertensive cardiovascular remodeling.

Precision gene editing technologies based on CRISPR/Cas9: a review / 生物工程学报

Gene editing technology is a genetic operation technology that can modify the DNA sequence at the genomic level. The precision gene editing technology based on CRISPR/Cas9 system is a gene editing technology that is easy to operate and widely used. Unlike the traditional CRISPR/Cas9 system, the precision gene editing technology can perform site-directed mutation of genes without DNA template. This review summarizes the recent development of precision gene editing technology based on CRISPR/Cas9, and prospects the challenges and opportunities of this technology.

Construction of a 10rolGLP-1-expressing glucose-lowing Saccharomyces cerevisiae by CRISPR/Cas9 technique / 生物工程学报

To develop a novel glucose-lowering biomedicine with potential benefits in the treatment of type 2 diabetes, we used the 10rolGLP-1 gene previously constructed in our laboratory and the CRISPR/Cas9 genome editing technique to create an engineered Saccharomyces cerevisiae strain. The gRNA expression vector pYES2-gRNA, the donor vector pNK1-L-PGK-10rolGLP-1-R and the Cas9 expression vector pGADT7-Cas9 were constructed and co-transformed into S. cerevisiae INVSc1 strain, with the PGK-10rolGLP-1 expressing unit specifically knocked in through homologous recombination. Finally, an S. cerevisiae strain highly expressing the 10rolGLP-1 with glucose-lowering activity was obtained. SDS-PAGE and Western blotting results confirmed that two recombinant strains of S. cerevisiae stably expressed the 10rolGLP-1 and exhibited the desired glucose-lowering property when orally administered to mice. Hypoglycemic experiment results showed that the recombinant hypoglycemic S. cerevisiae strain offered a highly hypoglycemic effect on the diabetic mouse model, and the blood glucose decline was adagio, which can avoid the dangerous consequences caused by rapid decline in blood glucose. Moreover, the body weight and other symptoms such as polyuria also improved significantly, indicating that the orally hypoglycemic S. cerevisiae strain that we constructed may develop into an effective, safe, economic, practical and ideal functional food for type 2 diabetes mellitus treatment.

Application of single base editing technique in pig genetic improvement: a review / 生物工程学报

Traditional pig breeding has a long cycle and high cost, and there is an urgent need to use new technologies to revitalize the pig breeding industry. The recently emerged CRISPR/Cas9 genome editing technique shows great potential in pig genetic improvement, and has since become a research hotspot. Base editor is a new base editing technology developed based on the CRISPR/Cas9 system, which can achieve targeted mutation of a single base. CRISPR/Cas9 technology is easy to operate and simple to design, but it can lead to DNA double strand breaks, unstable gene structures, and random insertion and deletion of genes, which greatly restricts the application of this technique. Different from CRISPR/Cas9 technique, the single base editing technique does not produce double strand breaks. Therefore, it has higher accuracy and safety for genome editing, and is expected to advance the pig genetic breeding applications. This review summarized the working principle and shortcomings of CRISPR/Cas9 technique, the development and advantages of single base editing, the principles and application characteristics of different base editors and their applications in pig genetic improvement, with the aim to facilitate genome editing-assisted genetic breeding of pig.

A novel CRISPR/Cas9-hLacI donor adapting system for dsDNA-templated gene editing / 生物工程学报

During the gene editing process mediated by CRISPR/Cas9, precise genome editing and gene knock-in can be achieved by the homologous recombination of double-stranded DNA (dsDNA) donor template. However, the low-efficiency of homologous recombination in eukaryotic cells hampers the development and application of this gene editing strategy. Here, we developed a novel CRISPR/Cas9-hLacI donor adapting system (DAS) to enhance the dsDNA-templated gene editing, taking the advantage of the specific binding of the LacI repressor protein and the LacO operator sequence derived for the Escherichia coli lactose operon. The codon-humanized LacI gene was fused as an adaptor to the Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus lugdunensis Cas9 (SlugCas9-HF) genes, and the LacO operator sequence was used as the aptamer and linked to the dsDNA donor template by PCR. The Cas9 nuclease activity after the fusion and the homology-directed repair (HDR) efficiency of the LacO-linked dsDNA template were firstly examined using surrogate reporter assays with the corresponding reporter vectors. The CRISPR/Cas9-hLacI DASs mediated genome precise editing were further checked, and we achieved a high efficiency up to 30.5% of precise editing at the VEGFA locus in HEK293T cells by using the CRISPR/SlugCas9-hLacI DAS. In summary, we developed a novel CRISPR/Cas9-hLacI DAS for dsDNA-templated gene editing, which enriches the CRISPR/Cas9-derived gene editing techniques and provides a novel tool for animal molecular design breeding researches.